CN113307853B - MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof - Google Patents

Abstract

The invention discloses a MYB transcription inhibitory factor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof. Belongs to the technical field of genetic engineering. According to the invention, the MYB transcription inhibiting factor participating in anthocyanin synthesis is screened and cloned through transcriptome data of Lycium ruthenicum Murr according to the annotation result and expression quantity difference of MYB transcription factors: LrMYB3, belonging to the R2R3 class of MYB transcription factors. Multiple sequence alignment and evolutionary tree analysis show that the gene belongs to the FaMYB1-like transcription repressing factor. qRT-PCR analysis showed that LrMYB3 was expressed in each tissue of Lycium ruthenicum and the expression level gradually increased as the fruit matured. Subcellular localization and transcriptional activity assay experiments showed that LrMYB3 is a transcription factor localized in the nucleus with no activation function.

Description

MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof.

Background

Lycium ruthenicum Murray (Lycium ruthenicum Murray.) belonging to Solanaceae (Solanaceae) Lycium barbarum (Lycium barbarum) is a spiny shrub, is rich in nutrition and can be used as both medicine and food, and is widely concerned by people due to the fact that the Lycium ruthenicum Murray is rich in a large amount of anthocyanin. The anthocyanin has stronger antioxidant activity, can help plants to resist stress environment, and is also beneficial to promoting human health.

Transcription factors, also known as trans-acting factors, generally refer to a class of proteins encoded by genes that specifically bind to the relevant cis-acting elements in the promoter region of the gene to activate or inhibit expression of the gene, thereby improving the adaptability of the plant to the environment. MYB transcription factors play an important role in anthocyanin metabolism regulation, and although research results report a plurality of MYB transcription factors positively regulating anthocyanin synthesis in Lycium ruthenicum, the MYB transcription factors inhibiting anthocyanin synthesis are not reported.

Therefore, how to provide a MYB transcription inhibitor related to anthocyanin synthesis of Lycium ruthenicum Murr is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a MYB transcription inhibitor LrMYB3 related to anthocyanin synthesis of Lycium ruthenicum Murr and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

an MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin, the amino acid sequence of which is shown in SEQ ID No. 2.

The invention also provides a MYB transcription inhibitor LrMYB3 gene related to anthocyanin synthesis of Lycium ruthenicum Murr, and the nucleotide sequence of the gene is shown in SEQ ID No. 1.

The invention also provides a recombinant vector containing the transcription inhibitor LrMYB3 gene.

The invention also provides a recombinant strain containing the transcription inhibitor LrMYB3 gene or the recombinant vector.

The invention also provides application of the transcription inhibitor LrMYB3 or the gene in Chinese wolfberry breeding.

Compared with the prior art, the invention screens and clones MYB transcription inhibiting factors participating in anthocyanin synthesis according to annotation results and expression quantity difference of MYB transcription factors through transcriptome data of lycium ruthenicum by the aid of the myB transcription inhibiting factors LrMYB3 related to anthocyanin synthesis of lycium ruthenicum and application of the myB transcription inhibiting factors: LrMYB3, wherein the gene cDNA thereof is 534bp in total and codes 177 amino acids; analysis by protein sequence databases showed that it contains 2 conserved MYB-DNA-binding domains belonging to the R2R3 class of MYB transcription factors. Multiple sequence alignment and evolutionary tree analysis show that MYB transcription inhibitors related to anthocyanin synthesis in other species are clustered, and belong to FaMYB1-like transcription inhibitors.

qRT-PCR analysis showed that LrMYB3 is expressed in various tissues of Lycium ruthenicum Murr, and the expression level gradually increases with the ripening of the fruit. Subcellular localization and transcriptional activity assay experiments showed that LrMYB3 is a transcription factor localized in the nucleus with no activation function.

Constructing a plant overexpression vector, and obtaining LrMYB3 transgenic tobacco and Arabidopsis strains through agrobacterium-mediated genetic transformation, wherein the result shows that the color of the corolla of the transgenic tobacco is lighter than that of a wild type, the corolla of the transgenic tobacco is light pink to white with little pigment accumulation, and qRT-PCR shows that the expression level of most structural genes related to anthocyanin synthesis is lower than that of the wild type, particularly structural genes NtDFR, NtANS and NtUFGT at the late stage of the anthocyanin synthesis path. Similar results are also obtained in transgenic arabidopsis, the seed coat of T1 generation seeds is lighter than that of a wild type, the seeds are light brown, no pigment is accumulated at the top of leaves and stems of seedlings under the stress of 6% of sucrose concentration, the wild type has partial pigment accumulation, and qRT-PCR analysis shows that the expression level of related structural genes of an arabidopsis anthocyanin synthesis pathway is obviously lower than that of the wild type.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the result of gel electrophoresis of the target gene LrMYB3 provided by the invention.

FIG. 2 is a drawing showing the hydropathic and hydrophobic analysis chart of LrMYB3 protein provided by the invention.

FIG. 3 is a diagram of the transmembrane domain of LrMYB3 protein provided by the invention.

FIG. 4 is a diagram showing the prediction of the LrMYB3 signal peptide provided by the invention.

FIG. 5 is a diagram showing the prediction of the tertiary structure of the protein according to the present invention.

FIG. 6 is a phylogenetic tree analysis of the LrMYB3 protein provided by the invention and MYB proteins in other species associated with anthocyanin synthesis pathways.

FIG. 7 is a multiple sequence alignment of the amino acid sequence of LrMYB3 provided herein with similar MYB proteins in other species.

FIG. 8 is a PCR identification of the LrMYB3 subcellular localization carrier liquid provided by the invention.

FIG. 9 the accompanying drawing is a subcellular localization map of LrMYB3 protein provided by the invention, wherein GFP: GFP fluorescence; DAPI: a nucleic acid dye; bright: bright field; merged: superposition of bright field, green fluorescence and blue fluorescence.

FIG. 10 is a diagram of an analysis of the transcriptional activity of LrMYB3 provided by the present invention, wherein the sequence from left to right is: one-lacking medium, three-lacking medium and three-lacking medium coated with X-alpha-Gal staining solution.

FIG. 11 is a graph showing the expression level analysis of LrMYB3 in black fruit and gingko fruit of Lycium barbarum provided by the present invention.

FIG. 12 is a diagram of transgenic Arabidopsis thaliana T0 generation seed screening provided by the present invention.

FIG. 13 is a representative seed map of wild type and transgenic Arabidopsis thaliana T1 provided by the present invention.

FIG. 14 is a diagram of 6% sucrose stress Arabidopsis seedlings provided by the present invention.

FIG. 15 is a diagram showing the expression levels of structural genes in anthocyanin synthesis provided by the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin and application thereof.

Some of the reagents and starting materials referred to in the examples are as follows:

(1) plant material: lycium ruthenicum Murr and Lycium ginkgo fruit collected from Lycium ruthenicum Murr resource nursery of Ningxia agriculture and forestry academy of sciences of Ningxia Hui nationality; common tobacco, Nicotiana benthamiana and Arabidopsis thaliana Col-0 stored in the experiment.

(2) Carrier: cloning vector 5minTM TA/Blunt-Zero Cloning Kit was purchased from Nanjing Novophilin Biotech GmbH; subcellular localization vector P1300-GFP, yeast PGBKT7 vector, vector PCM 1307.

(3) The strain is as follows: escherichia coli DH5 α available from Biotech, Inc., of Beijing Ongchongke; yeast AH109, Agrobacterium GV 3101.

The main reagents are shown in table 1 below:

TABLE 1

Solution and medium formulations:

enzymolysis liquid: a20 mM MES (pH 5.7) solution to which 1.5% (wt/vol) cellulase R10, 0.4% (wt/vol) macerase R10, 0.4M mannitol and 20mM KCl were added was placed in a thermostatic waterbath pan at 55 ℃ in a water bath for 10min to inactivate DNase and protease and improve the solubility of the enzymes. The heated enzyme solution was taken out and cooled to room temperature (25 ℃ C.), and 10mM CaCl was added₂And 0.1% BSA. The enzyme solution is ready for use.

WI solution: 0.5M mannitol and 20mM KCl were added to a solution of 4mM MES (pH 5.7). The prepared solution can be stored at room temperature (22-25 ℃).

W5 solution: 154mM NaCl, 125mM CaCl₂And 5mM KCl was added to a 2mM MES (pH 5.7) solution. The prepared solution can be stored at room temperature.

MMG solution: 0.4M mannitol and 15mM MgCl₂Adding into 4mM MES (pH 5.7) solution. The prepared solution can be stored at room temperature.

PEG-Ca transfection solution: 0.2M mannitol and 100mM CaCl₂Is added to ddH₂Completely dissolving O in 20-40% (wt/vol) PEG4000 solution.

10 × TE: 0.1M Tris-HCl 10mL, 10mM EDTA 2mL, water to 100 mL. Sterilizing at 121 deg.C for 20 min.

10 × LiAC: 1M LiAC (3.3 g of anhydrous lithium acetate was weighed out and dissolved in 40mL of water, adjusted to pH 7.5, made up to 50mL, filtered and sterilized).

1 × TE/LiAC: 0.1mL of 10 XTE and 0.1mL of 10 LiAC, and water was added to 1 mL.

40% PEG conversion solution: 0.8mL of 50% PEG solution, 0.1mL of 10 XTE, and 0.1mL of 10 LiAC.

YPAD medium: 10g/L yeast extract, 20g/L peptone, 50mL 40% glucose solution (115 ℃, sterilized alone for 15 minutes), 20g/L agar (solid medium), 15mL 0.2% Adenine solution (added when the medium is cooled to about 60 ℃), and sterilized at 121 ℃ for 20 minutes.

Defect culture medium: nitrogen base medium 26.7g/L, Amino acid mix Xg/L (kit, according to the volume of the preparation medium added corresponding amount), adjusting the pH value to 5.8, adding agar 20g/L, 121 ℃, sterilizing for 15 minutes. When the culture medium is cooled to about 60 ℃, 15mL of 0.2% Adenine solution and 50mL of 40% glucose solution are added.

LB culture medium: 10g/L sodium chloride, 5g/L yeast extract, 10g/L tryptone, 12.5g/L agar (solid), autoclaving at 121 ℃ for 20 min.

YEB Medium: beef extract 5g/L, peptone 5g/L, yeast extract 1g/L, sucrose 5g/L, MgSO₄·7H₂O0.5 g/L, agar (solid) 12.5g/L, autoclave sterilization at 121 ℃ for 20 min.

Immersing a dye solution: the MS powder is 2.215g/L, sucrose is 30g/L, acetosyringone is 200 mu mol/L (the medium is added after being cooled to about 60 ℃), the PH is adjusted to 5.2, and autoclaving is carried out at 121 ℃ for 20 min.

Co-culture medium: MS powder 4.43g/L, sucrose 30g/L, 6-BA 1mg/L, NAA 0.1mg/L, agar 7g/L, acetosyringone 200. mu. mol/L, adjusting pH to 5.8, and autoclaving at 121 deg.C for 20 min.

Induction differentiation culture medium: MS powder 4.43g/L, sucrose 30g/L, 6-BA 1mg/L, NAA 0.1mg/L, agar 7g/L, adjusting pH to 5.8, and autoclaving at 121 deg.C for 20 min. When the culture medium is cooled to be not too hot, 5mg/L of hygromycin and 300 mg/L of cef are added.

Rooting culture medium: MS powder 4.43g/L, sucrose 30g/L, adjusting pH to 5.8, autoclaving at 121 deg.C for 20min, and adding hygromycin 5mg/L and cef300 mg/L when the culture medium is cooled to no scald.

The arabidopsis inflorescence infection liquid formula comprises the following components: 2.215g/L MS powder, 2% sucrose, sterilizing at 121 ℃ for 20min, cooling the culture medium to room temperature, and adding Silwet solution with the final concentration of 0.02% -0.03%.

Here, the description is omitted.

Example 1

The extraction of DNA and the extraction of RNA are carried out by a conventional method according to the instruction of a kit.

Synthesis of Single-stranded cDNA: single-stranded cDNA synthesis was performed using the total RNA of Lycium ruthenicum fruit as a template and the experimental procedures given by the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) of Takara.

Cloning of the LrMYB3 gene: the sequences of the amplification primers are shown in Table 2, and the primers were diluted to 10. mu.M, and PCR amplification reaction was carried out using the obtained cDNA as a template, and the reaction system is shown in Table 3 below.

TABLE 2

TABLE 3

And after amplification is finished, taking out the PCR product, carrying out 1% agarose gel electrophoresis detection, carrying out electrophoresis for about 7min, observing under an ultraviolet gel imager, cutting a gel block of the PCR product, placing the cut gel block into a 2mL centrifuge tube if a target band is single and the size is correct, and carrying out gel recovery by using the operation steps of the Tiangen general DNA purification recovery kit.

Connecting and transforming Escherichia coli

The gene fragment of the recovered rubber product LrMYB3 is connected with a T cloning vector, and the reaction system is shown in the following table 4:

TABLE 4

After the reaction, the centrifuge tube was placed on ice, and E.coli DH 5. alpha. was transformed by heat shock method, as follows:

(1) the ligation product was added to a 1.5mL centrifuge tube containing 50. mu.l of E.coli competent DH 5. alpha. and gently whipped rapidly and gently using a pipette gun and mixed well and allowed to stand on ice for 30 min.

(2) After standing, the centrifuge tube was placed in a 42 ℃ water bath, heat-shocked for 1min, and immediately placed on ice for 2 min. Then, 1mL of LB liquid medium containing no antibiotic was added thereto, and the mixture was shaken in a shaker at 37 ℃ at 200rpm for about 1 hour.

(3) Taking out, centrifuging at 5500rpm for 2min, discarding most of supernatant in a clean bench, mixing the rest bacteria liquid, spreading on LB solid culture medium containing corresponding antibiotics, and placing in a 37 deg.C incubator overnight.

(4) The next day, the plates were removed, single colonies were picked on a clean bench into LB (antibiotic containing) liquid medium, which had been added to 1mL, and shaken in a shaker at 37 ℃ for 1-2 h. Colony PCR was performed using the universal primers M13-F and M13-R of the T cloning vector as identified in Table 5 below:

TABLE 5

Extraction of plasmid DNA from Escherichia coli

And (3) carrying out agarose gel electrophoresis on the colony PCR product, sucking 200 mu l of positive bacteria liquid with correct band size, sending the positive bacteria liquid to a company Limited in Biotechnology engineering (Shanghai) for sequencing, comparing a sequencing result with a target gene sequence, sucking 20 mu l of bacteria liquid into 10ml of LB liquid culture medium (containing antibiotics) after no errors, and carrying out overnight culture in a shaker at 37 ℃ at 200 rpm.

The next day, after the bacterial liquid has a certain concentration, taking out, sucking 1mL of bacterial liquid into a 1.5mL centrifuge tube in an ultra-clean bench, adding glycerol, mixing uniformly, and storing in a refrigerator at-80 ℃. And extracting plasmids from the residual bacterial liquid according to the steps given by the plasmid miniextraction kit of the Tiangen.

Example 2

The open reading frame and deduced amino acid sequence of the LrMYB3 transcription factor were searched by an ORF Finder (https:// www.ncbi.nlm.nih.gov/orffinder /) online search tool at NCBI. The amino acid composition of the transcription factor, the molecular weight of the protein, the theoretical isoelectric point and the stability were predicted by using the online software ProtParam (http:// web. expasy. org/ProtParam /). The hydrophobicity and charge distribution of the protein were analyzed using the online software ProtSca le (https:// web. expasy. org/protscale /). The transmembrane domain of the LrMYB3 protein was analyzed using TMHMM 2.0(http:// www.cbs.dtu.dk/services/TMHMM) software. Cell-PLoc 2.0(http:// www.cbs.dtu.dk/services/Sign alP) was used to predict the signal peptide of the protein. The subcellular localization of the protein was predicted using Wolf Psort (https:// www.genscript.com/Wolf-ptort. htmL) and online software (https:// www.csbio.sjtu.edu.cn/biooil/Cell-PLoc-2 /). Secondary and tertiary structure predictions were made for the protein using SOPMA (https:// NPSA-prabi. ibcp. fr/cgi-bin/nps a _ auto. page ═ NPSA/NPSA _ SOPMA. html) and SWISS MODEL (https:// swis smodel. expasy. org /), respectively. Conserved domains were analyzed using SMART (http:// smart.embl-heidelberg.de/SMART/set _ mode.cgi. LrMYB3 was aligned to homologous sequences from other species using DNAMAN 8.0, and phylogenetic trees were constructed using the Neighbor-Joining method (NJ) using MEGA7.0 software.

Construction of recombinant vectors:

cloning of homologous recombination fragment of target gene

Using the cDNA solution obtained in the above example as a template, the corresponding primers in Table 2 were used to amplify the homologous recombination fragments of LrMYB3-GFP, and the amplification system is shown in Table 6 below:

TABLE 6

And (4) carrying out agarose gel electrophoresis detection and gel recovery on the PCR product, and storing at-20 ℃ for later use.

Vector digestion reaction

The subcellular localization vector was double-digested with the cleavage sites BamHI and XbaI using a laboratory-stored pCAMBIA 1300-GFP. The yeast hybrid vector was double-digested with the cleavage sites EcoRI and BamHI using PGBKT7 stored in this laboratory. The cleavage system is shown in Table 7 below:

TABLE 7

And (4) carrying out agarose electrophoresis on the enzyme digestion product to verify whether the vector is cut open or not and whether the cut band is the expected size or not so as to ensure the accuracy of enzyme digestion. Cutting off the carrier gel block after the carrier gel block is ensured to be correct, recovering by using a universal DNA purification recovery kit, and storing the recovered carrier at-20 ℃ for later use.

Homologous recombination connecting fragment and vector

And (3) carrying out homologous recombination on the cloned target fragment with the homologous arm and a cut corresponding vector to construct a subcellular localization vector with LrMYB3-GFP and a PGBKT7 yeast recombinant BD vector with GAL4BD-LrMYB 3.

The homologous recombination ligation reaction system is shown in Table 8 below:

TABLE 8

Recombinant vector transformation of Escherichia coli DH5 alpha

And (3) carrying out the following steps on the constructed subcellular localization vector with the target fragment: LrMYB3-GFP and PGBKT7 vector with target fragment: GAL4BD-LrMYB3 was removed from the PCR machine, immediately placed on ice, and transformed into E.coli DH 5. alpha. in the same manner as in the above examples. And on the next day, carrying out PCR identification and sequencing on the conventional bacterial colonies after the bacterial colonies grow out, and confirming that the vector is successfully constructed.

Plasmid miniextraction was performed on positive bacterial fluid of GAL4BD-LrMYB3 (see kit instructions, general treatment).

Adding 100 mu l of positive bacterial liquid of LrMYB3-GFP into LB (including kanamycin) liquid culture medium containing 200mL, shaking for 24h, and after the bacterial liquid presents a certain concentration, carrying out plasmid large-extraction by using a Tiangen endotoxin-free plasmid large-extraction kit to obtain high-concentration and high-purity plasmids for subsequent protoplast transformation experiments.

Extraction of protoplasts

(1) Cutting off tender green, thick and well-grown tobacco leaves growing for 3-4 weeks (usually having 5-7 true leaves), placing on clean filter paper, immediately dipping a small amount of carborundum to gently and quickly rub the lower epidermis of the leaves until the leaves are smooth and have slight tissue fluid exudation, cutting the leaves into small blocks of 5 x 5mm by using a sharp blade, immediately and quickly and gently spreading the small blocks in prepared fresh enzymatic hydrolysate (so that the side with the lower epidermis ground away faces downwards) so that the leaves can fully contact the enzymatic hydrolysate to be fully digested.

(2) Wrapping the culture dish filled with the enzymolysis liquid by using tinfoil, and placing the culture dish in a constant-temperature shaking table at 37 ℃ for dark shaking culture for 2-3 h. During this period, the digestion was observed every 1h, and the enzyme solution was gently swirled and gradually turned green, indicating that the protoplasts had begun to be released.

(3) After the enzymatic solution had turned to a very concentrated green color and the leaves were relatively transparent, the undigested leaves were gently removed with forceps, 20. mu.l was pipetted onto a glass slide, and the protoplast release was examined microscopically.

(4) To determine the quality and concentration of protoplasts for subsequent experiments, the lysed solution was gently transferred to a 50mL flat-bottomed centrifuge tube and the protoplast solution was diluted with an equal volume of W5 solution. Centrifuging for 2min at 100g, removing the supernatant as much as possible, adding about 3-5 mL of W5 solution, gently rotating the centrifuge tube, and re-suspending the protoplast precipitated at the bottom of the tube.

(5) Aspirating 20. mu.l of the resuspended protoplast solution into a cell counter to observe the concentration of the protoplasts, which can be observed under a 100 Xmicroscope at 2X 10⁵And about one. The protoplast solution was allowed to stand on ice for 30 min.

(6) Centrifuge tubes at 100g for 1min, remove supernatant (do not contact protoplasts as much as possible), add 2mL of MMG solution, gently rotate the tube, resuspend and mix protoplasts. Placed on ice for subsequent transformation experiments.

Protoplast transformation step (DNA-PEG-Ca transformation method):

(1) add 10. mu.l (10-20. mu.g) of recombinant subcellular localization plasmid DNA to a 2mL centrifuge tube, add 100. mu.l of protoplast resuspended and mixed in MMG solution, mix by gently rotating the centrifuge tube.

(2) Add 110. mu.l fresh PEG-CaCl to the centrifuge tube₂The solution was mixed well by tapping the centrifuge tube wall. Incubate at room temperature for 6 min. After the incubation was complete, 440. mu.l of W5 solution was added and the tube was gently inverted to terminate the transfection process. Centrifuge at 100g for 2min and remove supernatant. Adding 1M W5 solution to wash protoplast, centrifuging for 2min at 100g, and removing supernatant.

(3) The surface of the 6-well cell culture dish was coated with 0.1% BSA (sterilized) solution to prevent the protoplasts from adhering to the surface of the dish. Protoplasts resuspended in WI solution were gently transferred to a petri dish, wrapped in tinfoil and incubated overnight at 25 ℃ in the dark.

Microscopic observation of GFP fluorescence signals

(1) The overnight cultured protoplasts were transferred from the cell culture dish to a 2mL centrifuge tube, centrifuged at 100g for 2min, the majority of the supernatant was removed, and the protoplasts were resuspended in a gently swirled centrifuge tube.

(2) The transformed protoplast solution was pipetted onto a slide and the GFP fluorescence signal was observed under a fluorescence microscope.

(3) To determine the location of the nuclei, the protoplasts were subjected to DAPI staining. The protoplast solution and the ready-to-use DAPI solution with the same volume are mixed evenly, the mixture is dyed on ice for 5-10 min in a dark place, the protoplast is diluted by 1 Xphosphate buffer solution PBS, and the dyeing is stopped. Protoplasts stained with DAPI were pipetted onto a glass slide and observed under a fluorescent microscope for a blue fluorescence signal.

Yeast transformation

(1) Yeast AH109 strain was streaked on YPAD medium and cultured in an inverted state at 28 ℃ for 3 days in a constant temperature incubator. After the bacterial colony grows out, picking a single bacterial colony in an ultraclean workbench to a conical flask containing 5mL of YPAD liquid culture medium, and placing the conical flask in a constant-temperature shaking table at 28 ℃ for overnight culture.

(2) In the morning of the next day, 10 times the volume of fresh YPAD liquid medium was added to the flask, and the culture was continued until the OD was 0.3-0.6. When the culture medium is cultured to a proper concentration, the culture medium is taken out, poured into a 50mL centrifuge tube in an ultra-clean bench and centrifuged at 5000rpm at room temperature for 5 min.

(3) The liquid in the centrifuge tube was decanted, resuspended in 20mL sterile water, and centrifuged at 5000rpm for 5min at room temperature.

(4) A1 XTE/LiAC solution was prepared as required (ready to use). The liquid in the centrifuge tube was decanted, washed with 1mL of 1 XTE/LiAC solution into a 2mL centrifuge tube, and centrifuged at room temperature for 3 min. Resuspend with 300. mu.l to 500. mu.l of 1 XTE/LiAC solution. Thus, yeast competence is completed.

(5) To a 2mL centrifuge tube were added 5. mu.l of plasmid DNA, 10. mu.l of salmon sperm DNA (boiled in boiling water for 10min before use and placed on ice) and 100. mu.l of yeast competent in that order, and mixed well. Shaking and culturing for 30min at 28 deg.C. Then placing the centrifuge tube in a water bath kettle at 42 ℃ for 15min, and uniformly mixing the centrifuge tube once.

(6) The centrifuge tube is taken out, the yeast sediment is collected by instant centrifugation at room temperature, and then the yeast sediment is resuspended by 1mL of sterile water and centrifuged for 1min at 5000 rpm. The supernatant was discarded, 100. mu.l of sterile water (depending on the concentration) was added for resuspension, 50. mu.l of the resuspension solution was applied to a lacking medium (SD/-Trp), and the resulting mixture was cultured in an inverted state in a constant temperature incubator at 28 ℃ for 3 days.

(7) Yeast dotting: the single colonies that grew out were picked up in 30. mu.l of sterile water, resuspended and 2. mu.l pipetted onto yeast-minus (SD/-Trp) and yeast-minus (SD/-Trp/-His/-Ade) media, respectively. The cells were photographed after inverted culture in a constant temperature incubator at 28 ℃ for 3 days.

Real-time fluorescent quantitative PCR analysis

Based on the cloned LrMYB3 gene sequence, the cDNA solutions of the tissues obtained according to the method of example 1 were diluted 4-fold using the primer sequences of Table 2, and the procedures of quantitative PCR detection kit using Vazyme high-specificity SYBR dye method were performed in Bio-Rad CFX96^TMThe PCR reaction was carried out in a real-time PCR detection system as follows:

TABLE 9

The relative expression level of the gene was calculated from the qPCR results using the 2- Δ Δ Ct method, with 3 biological replicates per experiment.

The results show that:

and carrying out PCR amplification by taking the cDNA as a template to obtain a band with the size of 534bp, connecting a T cloning vector, sequencing by using a universal primer, and determining a sequencing result to be basically consistent with a gene sequence obtained by a transcriptome to be a target gene fragment. The results of gel electrophoresis are shown in FIG. 1:

bioinformatic analysis of LrMYB3 gene: the gene Open Reading Frame (ORF) of LrMYB3 is 534bp (ATGAGAAAACCTTGTTGTGATCATAACAAGGAGGAAATGCAAAGAGGAGCTTGGTCTAAACAAGAAGACCAAAAACTCATTGATTATATCACTAAACATGGTGAAGGTTGCTGGAGAAACTTACCTAAAGCTGCTGGTCTGCTTCGCTGCGGAAAAAGTTGCAGGCTGAGATGGATTAATTATCTTAGTCCAAATCTAAAAAGAGGCAATTTTTCTGAGGATGAAGATGATCTCATCATCAAGCTTCATGCTCTACTTGGCAACAGATGGTCCCTAATAGCTGGAAGATTACCGAGGAGAACTGATAATGAAGTGAAGAATTATTGGAATTCCCATTTGAGAAGAAAACTAATAAAAATGGGAATCGATTCAAAAAATCATAGGATTTCTCATTATCTTCATATAAAAAGGCTTGAATTTTTGCCAAAAAATAACACAAGAGAAAATGATGGAGTAATATCTGATACTGCAAGTTCTTGTGCAGATGGTCAACAAATTACAAGTTCATTGCCTGATCTCAATTCACTTCCATAG, as shown in SEQ ID No.1), and codes 177 amino acids (MRKPCCDHNKEEMQRGAWSKQEDQKLIDYITKHGEGCWRNLPKAAGLLRCGKSCRLRWINYLSPNLKRGNFSEDEDDLIIKLHALLGNRWSLIAGRLPRRTDNEVKNYWNSHLRRKLIKMGIDSKNHRISHYLHIKRLEFLPKNNTRENDGVISDTASSCADGQQITSSLPDLNSLP as shown in SEQ ID No. 2).

Hydropathic and hydrophobic analysis, transmembrane analysis and tertiary structure prediction of the protein encoded by LrMYB 3:

analysis using the online software ProtParam and ProtScale showed a theoretical molecular weight of 20533.44D, a theoretical isoelectric point of 9.48, and a protein destabilization coefficient of 54.03, indicating that the protein is a labile protein with a mean hydrophilicity (GRAVY) of-0.858 (table 10), indicating that the protein is a hydrophilic protein (fig. 2).

Watch 10

Neither the transmembrane region nor the signal peptide was predicted for the LrMYB3 protein (fig. 3, fig. 4) using TMHMM and SignalP 4.1, indicating that the protein is a non-transmembrane protein and a non-secreted protein.

The secondary structure of the LrMYB3 protein was predicted using SOPMA to include 30.51% alpha helix (alpha-helix), 7.91% beta turn (beta-turn), 10.17% beta sheets and 51.41% random coil (table 11).

TABLE 11

The similarity of the tertiary structure MODEL of LrMYB3 protein constructed by SWISS-MODEL to WER transcription factors was 63.06% (see FIG. 5).

(3) The conserved domain analysis of the protein by using SMART shows that the LrMYB3 protein has two conserved domains (repeated R sequences) at the N terminal, the amino acid sequence from 14 to 64 is the conserved domain of R2, and the amino acid sequence from 67 to 115 is the conserved domain of R3, so that the LrMYB3 protein belongs to the subfamily R2R 3.

The subcellular localization prediction of LrMYB3 was performed using the Cell-PLoc 2.0 online tool and the results showed localization in the nucleus.

The LrMYB3 protein is subjected to Clustalw comparison with MYB protein sequences related to anthocyanin synthesis routes in species such as arabidopsis, apples, grapes and strawberries by using software MEGA-X, the comparison result is analyzed by using a Neighbor-Joining method, Bootstrap is set to be 1000, and a phylogenetic tree is constructed. Analysis showed that LrMYB3 was associated with FaMYB1-like transcriptional repressors of the R2R3-MYB subgroup, such as petunia PhMYB27, grape VvMYBC2-L3, peach PpMYB18, strawberry FcMYB1, etc (FIG. 6). Therefore, it can be presumed that LrMYB3 belongs to the R2R3-MYB class of transcription repressors, and that the FaMYB1-like class of transcription repressors in other species with which it is grouped have similar functions.

Multiple sequence alignment of LrMYB3 proteins

Multiple sequence alignment analysis of the amino acid sequence of LrMYB3 with more homologous MYB proteins in other species using DNAMAN 8.0 software indicated that LrMYB3 has two DNA binding domains, R2 and R3, at its N-terminus, indicating that LrMYB3 is a MYB transcription factor of the R2R3-MYB class and that LrMYB3 has an essential motif that binds to bHLH transcription factors: [ D/E ] Lx2[ R/K ] x3Lx6Lx 3R. In addition, two inhibitory motifs, namely a C1 motif (LIsrGIDPxT/SHRxI/L) and a C2 motif (DLNxxP/LxLxLxLx), were also present at the C-terminus of LrMYB3, suggesting that the protein may exert a transcriptional repression function, thereby negatively regulating anthocyanin accumulation (FIG. 7).

Subcellular localization analysis of LrMYB3 protein

The recombinant vector LrMYB3-GFP (FIG. 8) and the empty vector P1300-GFP as a control were transferred into tobacco protoplasts under the drive of a CaMV35S constitutive promoter. The control vector was observed to distribute green fluorescence throughout the cells under a fluorescent microscope, whereas LrMYB3-GFP only emitted a strong fluorescent signal in the nucleus (FIG. 9). It is known that transcription factors are localized in the nucleus and all function as transcription regulators in the nucleus to exert their regulatory functions.

Transcriptional Activity assay for LrMYB3

To verify whether LrMYB3 is transcriptionally active, a PGBKT7 recombinant plasmid containing LrMYB3 was constructed, with empty PGBKT7 as a negative control and PGBKT7 linked with LrAN11 (transcriptionally active) as a positive control. The results showed that both control and experimental groups grew normally on a single absence of medium (SD/-Trp), indicating successful transfer of the plasmid into yeast. Whereas none of the growth on triple-gap medium (SD/-Trp/-His/-Ade) except the positive control, showed that none of LrMYB3 had transcriptional activation activity and also did not turn blue in the triple-gap medium coated with X- α -Gal stain, whereas the positive control had a lighter blue color (FIG. 10). Therefore, we preliminarily concluded that LrMYB3 has no transcriptional activation activity in yeast cells and hypothesize that transcription factors need to bind to other transcription factors to form complexes to regulate gene expression.

Expression analysis of LrMYB3 in Black fruit and Ginkgo biloba and Lycium barbarum fruit

In order to verify the expression level of LrMYB3 obtained from transcriptome data, the expression level of LrMYB3 was analyzed by qRT-PCR in different developmental stages (S1-S5) of the Lycium barbarum fruit and the Lycium barbarum fruit. The result shows that the expression level of LrMYB3 in Lycium ruthenicum Murr is significantly higher than that of Lycium barbarum, and the expression level of LrMYB3 also shows a gradual rising trend along with the increase of anthocyanin content in the fruit ripening process, but the expression level begins to decrease at the S5 stage of full fruit ripening. However, in the fruit of lycium barbarum l, LrMYB3 was hardly expressed from the S1 stage where the fruit began to develop to the S5 stage where the fruit was fully ripe (fig. 11).

Tissue-specific expression analysis of LrMYB3 in Lycium ruthenicum Murr

And (3) carrying out qRT-PCR analysis by taking cDNA of different tissues of the lycium ruthenicum as a template. The results show that the LrMYB3 is expressed in stems, leaves, flowers and fruits, no expression specificity is realized, and the expression level of LrMYB3 in leaves and fruits is highest.

qRT-PCR analysis shows that the expression level of LrMYB3 is positively correlated with the accumulation of anthocyanin, and has high-level expression when the fruits begin to accumulate anthocyanin greatly, which shows that LrMYB3 is activated when the anthocyanin begins to accumulate, and shows that R3-MYB transcription inhibitor can be activated when the anthocyanin begins to synthesize so as to provide feedback regulation, and plays an important role in balancing the accumulation of anthocyanin.

Example 3

Construction of plant overexpression recombinant vector

Cloning of homologous recombination fragments of target genes:

using the cDNA solution obtained in the above example as a template, the corresponding primers in Table 12 were used to perform homologous recombination fragment amplification of plant overexpression vectors, and the amplification system, PCR product detection and recovery were as described in the above example.

TABLE 12

Vector digestion reaction

Carrying out double digestion on the vector PCM1307 by using two restriction enzymes of Xba I and Kpn I; the vector pZYB9-pEAQ-HT was double-digested with two restriction enzymes Sma I and StuI. The cleavage system and the recovery of the product were the same as in the above example.

The homologous recombination connecting fragment and the vector, the transformation of Escherichia coli DH5 alpha, the PCR identification of bacterial liquid and the plasmid extraction are the same as the above embodiment.

Transformation of Agrobacterium tumefaciens

The operation steps are as follows:

(1) the prepared competent Agrobacterium tumefaciens was taken out from-80 ℃ and placed on ice.

(2) Mu.l of recombinant plasmid and 100. mu.l of competent Agrobacterium were added to a pre-cooled 1.5mL centrifuge tube and allowed to stand on ice for 30 min.

(3) Placing the centrifuge tube into liquid nitrogen for quick freezing for 5min, taking out, placing into 37 deg.C water bath, heating in water bath for 5min, and standing on ice for 2 min.

(4) 1mL of LB liquid medium without antibiotics was added to the centrifuge tube in a clean bench and cultured in a shaker at 28 ℃ for about 4 h. The cells were removed, plated with LB plates (supplemented with kanamycin and rifampicin), and cultured in an inverted state at 28 ℃ for 2 days.

(5) After the colonies grew out, single colonies were picked up and shake-cultured overnight at 28 ℃ in LB-resistant liquid medium. The next day, the colonies were taken out for PCR verification. Storing the positive bacteria liquid in a refrigerator at-80 ℃.

Genetic transformation of Arabidopsis thaliana

Agrobacterium mediated arabidopsis inflorescence infection method

(1) A single colony of fresh agrobacterium is picked up and put into 1mL LB resistant liquid medium, and shake cultivation is carried out at 28 ℃ overnight. The next day, the cells were diluted 1:50 and cultured to OD₆₀₀Approximatively closing to 0.8-1.0, centrifuging at 5500rpm for 5min, removing supernatant, and resuspending to OD with infection solution₆₀₀≈0.8。

(2) Watering wild Arabidopsis plants thoroughly one day and night before infection, selecting Arabidopsis plants in flowering phase and with good growth condition for transformation, and cutting off all pods of seedlings before transformation. All inflorescences of Arabidopsis were infected in a beaker containing the bacterial solution for 30 s.

(3) Preserving moisture of infected plants by using a preservative film, and culturing for 24 hours in a greenhouse at 23 ℃ in the dark.

(4) After 24h of dark culture, the preservative film is removed and the culture is carried out under normal illumination conditions. In order to improve the transformation efficiency, the infection is repeated once after 10 days, and the seeds are normally cultured until the seeds are harvested after the infection is finished.

(5) Mature seeds of T0 generation are collected, and a resistance culture medium containing hygromycin is used for screening positive plants. And culturing the screened positive plants until T1 generation seeds are collected, and drying the seeds appropriately and storing the seeds for subsequent experiments.

Sugar stress of transgenic Arabidopsis

Sterilizing the dried Arabidopsis seeds in a super clean bench, sowing the seeds in 1/2MS with 3% sucrose concentration and 1/2MS with 6% sucrose concentration, vernalizing the seeds in a refrigerator at 4 ℃ for 3 days, and taking the seeds out of a greenhouse at 25 ℃ for normal culture after 3 days. And after 2-3 weeks, observing the accumulation of anthocyanin.

Anthocyanin content determination

(1) About 0.1g of the material to be determined is ground to a powder in liquid nitrogen and 600. mu.l of 1% HCl in methanol are added immediately. Shaken at 4 ℃ for 6 h.

(2) After 6h, the mixture was taken out, 400. mu.l of water and 400. mu.l of chloroform were added thereto, and the mixture was centrifuged at 14000rpm in a refrigerated centrifuge at 4 ℃ for 5 min.

(3) The upper phase solution with the dissolved anthocyanins was transferred to a clean 1.5mL centrifuge tube and 200. mu.l of absorbance was taken for A530nm and A657 nm. The anthocyanin content (mg/g) is calculated by using a formula, namely A530nm-0.33 xA 657 nm. Three biological replicates were performed per sample.

The results show that:

the obtained LrMYB3 transgenic T0 generation plants are harvested seeds and sown in 1/2MS culture medium containing 35mg/L hygromycin, and the positive plants have hygromycin resistance, so that the leaves are tender green, the growth and development are normal, and the growth vigor is good. While wild type arabidopsis thaliana grew slowly because it did not contain a resistance gene, and slow yellowing stopped growing (fig. 12). Growing in a culture medium for about 10 days, selecting plants with good growth state and large body size, transferring the plants into nutrient soil for culturing, performing PCR identification and sequencing after 2 weeks, continuing culturing after positive plants are determined, and collecting mature T1 generation seeds. As a result, as shown in FIG. 13, the seed coat of transgenic Arabidopsis thaliana of the T1 generation having the LrMYB3 gene was lighter in color and appeared light brown compared to wild type Arabidopsis thaliana, while the wild type seed coat was dark brown. The L rMYB3 transgenic strain is shown to have less procyanidine content, and the transcription factor is supposed to be possibly involved in inhibiting procyanidine accumulation in seed coats.

Research shows that sucrose can promote accumulation of anthocyanin, so that the arabidopsis thaliana is stressed by high-concentration sucrose, and the function of the gene in anthocyanin synthesis regulation is researched. According to the previous study, Arabidopsis thaliana accumulates anthocyanin at 6% sucrose concentration. Therefore, in order to verify whether the LrMYB3 can inhibit the accumulation of anthocyanin in Arabidopsis under sugar stress conditions, 3 transgenic positive plants (OE #1, OE #2 and OE #3) are respectively selected and subjected to Arabidopsis high sugar stress treatment. The results showed that wild type arabidopsis seedlings grown for 2-3 weeks on 1/2MS medium with 6% sucrose accumulated some of the pigments in the leaves, petioles and top part of the stem, whereas transgenic LrMYB3 plants had almost no accumulation of pigments (fig. 14).

The anthocyanin content of the wild type and the transgenic seedling line is measured, and the pigment content is consistent with the phenotypic change of the wild type and the transgenic seedling line.

The expression levels of LrMYB3 gene in transgenic plant and structural genes (AtCHS, AtCHI, AtDFR, AtANS and the like) in the synthesis pathway of arabidopsis anthocyanin are analyzed by utilizing qRT-PCR, and the expression level of the structural genes related to anthocyanin synthesis in transgenic plant is remarkably reduced, which shows that the transcription factor of LrMYB3 (figure 15) can inhibit anthocyanin accumulation of arabidopsis under high sugar stress.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims (5)

1. A MYB transcription inhibitor LrMYB3 related to synthesis of lycium ruthenicum anthocyanin is characterized in that the amino acid sequence is shown in SEQ ID No. 2.

2. The gene of MYB transcription inhibitor LrMYB3 related to anthocyanin synthesis of Lycium ruthenicum as claimed in claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1.

3. A recombinant vector comprising the transcription repressing factor LrMYB3 gene of claim 2.

4. A recombinant strain comprising the transcription repressing factor LrMYB3 gene of claim 2 or the recombinant vector of claim 3.

5. Use of the transcription repressing factor LrMYB3 as defined in claim 1 or the gene as defined in claim 2 in Lycium ruthenicum breeding.

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